Evolution of genus is accompanied by ecological diversi cation. The majority of species grow in open, sunny, rather dry sites in arid and moderately humid climates. However, Allium species have adapted for many other ecological niches. Classical approaches for the identi cation of Allium cultivars are based on morphological traits. The assessment of these traits is di cult and their evaluation can be subjective considering that most of these cultivars are closely related. Hence, this study of Internal Transcribed Spacer (ITS) sequencing and four barcoding regions, matK, rbcL, trnH-psbA, trnL and Inter Simple Sequence Repeats (ISSR) were researched in onion, Allium cepa L. (Alliaceae) collected from three different cultivation sites. The results established noticeable hereditary divergence among the three cultivars. In ITS and matK, BDUT 1453, BDUT 1454 and BDUT 1455 were independent of each other and formed three clusters. In rbcL, BDUT 1453 formed an independent cluster from the cluster of BDUT 1454 and BDUT 1455. But in trnH-psbA, BDUT 1454 formed an independent cluster and BDUT 1453 and BDUT 1455 were closely placed whereas trnL showed all the three forming a cluster wherein BDUT 1453 and BDUT 1454 were placed closely in a sub-cluster. In ISSR, BDUT 1454 and BDUT 1455 formed a single cluster and BDUT 1453 diverged from it. Even though the tested cultivars belong to the same species they showed genetic divergence among themselves.